Multiple Endocrine Neoplasia Type 2 (MEN2) is an autosomal dominant genetic disorder characterized by the association of endocrine tumors. MEN2 is classified into two subtypes, MEN2A and MEN2B, according to the affected organs. The most important clinical manifestation of MEN2 is the medullary thyroid carcinoma (MTC), occurring in more than 95% of patients. Mutations in the RET (REarranged during Transfection) gene are responsible for the MEN2 phenotype. The clinical course of MTC in patients with MEN2A is variable, and the disease progression is associated with codon-specific mutations in the RET gene. The codon 634 (exon 11) is the most affected, accounting for up to 85% of MEN2A cases. MEN2B is most commonly caused by the p.M918T mutation (exon 16). The American Thyroid Association (ATA) guideline classifies patients with the p.M918T mutation as highest risk for MTC development and recommends total thyroidectomy before the first year of life; patients with a mutation in codon 634 are classified as high risk, and thyroidectomy should be recommended before 5 years of age. Surgery is the only curative treatment for MTC. Patients with intrathyroidal tumor have a 10-year survival rate of 95.6%, whereas patients with regional stage disease or distant metastasis at diagnosis present overall survival rates of 75.5 and 40%, respectively. These data illustrate the need to advance the knowledge of the molecular mechanisms involved in the MTC pathogenesis, the cumulative knowledge regarding allows new perspectives for the therapy of metastatic disease. Recently, advances in molecular biology provided great leaps in genetic engineering technologies. CRISPR is a powerful tool for editing genomes and allows us to easily alter DNA sequences and modify gene function. In addition, recent advances in this technology make it possible to correct point mutations. Based on that, we intend to correct gene mutations by gene editing of TT (p.C634W) and MZC (p.M918T) cell lines using the CRISPR/Cas9 system and evaluate the phenotype of parental (mutated) and edited (non-mutated) cells, generating a study model for the disease; we hope to correct RET gene mutations from MTC derived cell lines and evaluate the phenotype of parental (mutated) and edited (non-mutated) cells, thus generating a model for the study of MEN2 by the CRISPR/Cas9 system. To monitor tumor development in vivo, we will perform xenotransplantation of MZ-CRC-1 and TT cell lines in zebrafish embryos, moreover cells edited by CRISPR-Cas9.
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